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Toxicity to terrestrial plants

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Fe(Na)HBED showed positive effects on plants. 

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Experimental data:

Soybean (Glycine max L. cv Stine 0480) plants were used in a growth chamber experiment to investigate the efficacy to supply iron (Fe) to plants with different chelates, i.e. HBED/57Fe3+ (Lucena, Hernández and Nadal, 2008). Seeds were germinated using a standard seed growing procedure in closed sterilised trays for 2 days after pre-treatment. A 2:1 (v:v) soil:sand mixture was used and a daily quantity of solution was applied to achieve 80 % of field capacity. The experiment consisted on 2 treatments with six different dosses and one control without iron. Treatments were applied 7 days after transplanting, whereby plants showed symptoms of chlorosis at this time point. Photodegradation and algae development was avoided by covering the pots with a dark plastic film. 57Fe was used to study the efficacy of chelates, ICP-MS (Inductively Coupled Plasma Mass Spectroscopy) was the method of choice for detection. The treatments were performed with 0, 1.7, 3.4, 8.4, 16.8, 25.1 and 41.9 µmol of 57Fe/kg soil. 70 mg/L of Fe in form of the chelate (HBED/57Fe3 +) were prepared and 25 mL of them were added to the centre of the pot together with the water necessary to achieve the required field capacity. After the application and every two days, the SPAD (Soil Plant Analysis Development) Index was determined. Sampling times were one and three weeks after treatment, the total test duration was 24 months. After the plant experiment, the soluble and available 57Fe in soils were determined. Means were compared using Duncan´s test in order to find significant differences due to the treatment and statistical analysis was done with SPSS 15.0. In conclusion, HBED/Fe3+ is a good chelate to correct Fe chlorosis. In comparison to another chelate, EDDHA/Fe3 +, the following findings are reported: EDDHA/FE3+ present a faster action than HBED/Fe3+. HBED/Fe3+ presents more long lasting effect than EDDHA/Fe3+.


Furthermore, an expert statement was written by Lucena (2012) in order to gather information on the possible toxic effects of NaFeHBED (Sodium salt of the ferric chelate of (N,N'-Di{2-hydroxybenzyl)ethylenediamine-NiN'-diacetic acid) to plants. Citation: “NaFeHBED is a salt that easily dissolves in water to yield the Na+ cation and the Fe-HBED- anion. Fe-HBED (the negative charge is omitted hereafter) is a chelate that presents very high stability (logKo = ~39, Ma et al., 1994). As consequence of this high stability, Fe-HBED is very inert in the environment. No substitution of Fe by other metal is possible nor has been experimentally observed (Lopez-Rayo et al., 2009). The organic moiety is also stabilized in the chelate impeding the direct interaction with living organism. Moreover chemical reduction of the Fe(lll) present in the chelate to Fe(ll) in aqueous media is not possible. However, in the presence of the Iron Chelate Reductase Enzyme (IRT1) the chelate can be reduced releasing Fe(ll) and the chelating agent HBED. The Fe(ll) can be taken by the plants by a specific transporter and the chelating agent will chelate more Fe(lll) from the soil. IRT1 is an enzyme that is present in plant root tips of strategy I plants (dicotyledonous and monocotyledonous non Poaceae plants) (Robinson et al., 1999) and it is upregulated, so it is activated during Fe deficiency but repressed on Fe sufficient media (Vert et al., 2002). As consequence FeHBED should not be toxic for plants when used in a wide concentration range even over the recommended doses. It is also important to note that during germination plants mostly use seed Fe, so the reduction mechanism is not active in the early stages, so an excess of Fe(ll) is not expected at all. Moreover seeds can be covered with (solid) chelates or complexes prior to their use in field in order to localize the chelate close to the emerging roots so they can begin to reduce and uptake iron as soon as they can. Chaney (1988) demonstrated that plants can utilize iron from Fe-HBED due to the reduction mechanism previously discussed. On that paper Chaney successfully used a concentration of 10 u.M Fe-HBED on two strategy I plant species: soybean and nutsedge. Corn (a Poaceae species) was not able to take iron from Fe-HBED. It was also demonstrated that using Fe-HBED in nutrient solution is the best way to control Fe delivery to the plant without any interference.

Lanquar et al. (2004), used Fe-HBED for Arabidopsis seedling growth and observed that there were no toxicity at all. Moreover its use was beneficial for the plant to resist Cd toxicity.

Also in works from Lucena and Chaney (2006 and 2007) 10, 20 and 50 µM Fe-HBED were used for normal plant growth and even 100 µM Fe-HBED in experiments to determine the reduction capacity of the plants.

Other authors (e.g. Werger et al., 2006) have use Fe-HBED for Fe nutrition studies with algae. They present data of different acquisition mechanism by algae and also they conclude "that iron acquisition from Fe3+-HBED might serve as an assay for an organisms' ability to access tightly complexed iron".

So far, it has not been observed ever any toxicity or negative effect on the development of seeds, seedling or plants grown using Fe-HBED as Fe source in a wide range of concentration.

Fe-HBED has been proposed as a fertilizer and soil experiments are been carried out and therefore tested in different crops (soybean, cucumber, strawberry, stone fruits...), always with positive effect on plant growth and Fe nutrition. Some experiments has been conducted of growth chamber and others on open fields. In Nadal et al., 2009, 100 µM of Fe-HBED was used in reductase experiments and 5 and 10 urn Fe-HBED to grow plants in hydroponics. A new paper will be published soon in Plant and Soil (Nadal et al., 2012) presenting experiments using a wide range of doses of the chelate applied to soil (the highest one equivalent to 125 kg product/Ha, 5 times larger than the highest recommended doses in the field and 10 times higher than the adequate doses found in our experiment) and there was no any toxicity symptoms nor growth reduction of the seedling or adult plants. Three year field experiments (two locations in Aragon, Spain) developed by our group on mature trees (paper in preparation) indicated that the plants responded adequately to normal and double doses of Fe-HBED. No toxic effects were observed nor in the trees nor in the surrounding vegetation that was allowed to grow between trees' rows. Application at the recommended doses (3 times) to strawberry commercial crops in Huelva (Spain) under fertigation provides also good plant response both on Fe nutrition and on plant development. No adverse effects were observed. Demonstration experiments (several locations) on young and mature plants, on citrus and fruit trees, comparing three different doses, have also indicated that there is not any plant damage, not only on the crops but also on the grass between rows where allowed to grow.

It is worthy to indicate that field applications are made focalize, close to the drippers in fertigation or near the tree, using highly concentrate solutions or even solids. Not even in these conditions, it has been observed negative effects in the plants directly growing over the application place respect the controls.

In conclusion, it can be stated that after 23 years of using Fe-HBED as iron source in plant nutrition experiments and after 6 years of growth chamber, commercial greenhouses and field experiments using Fe-HBED fertilizers there is no any record showing toxicity problems not in seed germination, seedling development nor on plant growth and production.”



The test substance is used as iron fertilizers, thus it remedy iron chlorosis in plants in calcareous soils at high pH values. The substance is expected to have a low potential for soil adsorption; based on its low logPow and logKoc. Furthermore, no bioaccumulation is expected and it is not very toxic to aquatic organisms.

The described experimental results obtained in soil and plants proved that there is no toxicity potential to seedlings and plants.

Based on the intrinsic substance properties and in accordance to REACH, Annex IX, Section 9.4, column 2, no further investigations for toxicity in terrestrial plants is intended and this endpoint can be waived.